Properties of the human muscle nicotinic receptor, and of the slow-channel myasthenic syndrome mutant  L221F, inferred from maximum likelihood fits

Abstract

The mechanisms that underlie activation of nicotinic receptors are investigated using human recombinant receptors, both wild type and receptors that contain the slow channel myasthenic syndrome mutation, epsilonL221F. The method uses the program HJCFIT, which fits the rate constants in a specified mechanism directly to a sequence of observed open and shut times by maximising the likelihood of the sequence with exact correction for missed events. A mechanism with two different binding sites was used. The rate constants that apply to the diliganded receptor (opening, shutting and total dissociation rates) were estimated robustly, being insensitive to the exact assumptions made during fitting, as expected from simulation studies. They are sufficient to predict the main physiological properties of the receptors. The epsilonL221F mutation causes an approximately 4-fold reduction in dissociation rate from diliganded receptors, and a smaller increase in opening rate and mean open time. These are sufficient to explain the approximately 6-fold slowing of decay of miniature synaptic currents seen in patients. The distinction between the two binding sites was less robust, the estimates of rate constants being dependent to some extent on assumptions, e.g. whether an extra short-lived shut state was included or whether the EC50 was constrained. The results suggest that the two binding sites differ by roughly 10-fold in the affinity of the shut receptor for ACh in the wild type, and that in the epsilonL221F mutation the lower affinity is increased so the sites become more similar.

Single-Channel Behavior of Heteromeric α1β Glycine Receptors: An Attempt to Detect a Conformational Change before the Channel Opens

“…We therefore proceeded to test whether adding extra shut states to the unconstrained form of scheme 1 helped account for observed channel behavior. Only a slight improvement was detected when a single shut state was added in a position distal to either A 3 R or A 3 R* (Salamone et al, 1999;Hatton et al, 2003) (data not shown). Adding a distal shut state to scheme 1 or scheme 2 (while keeping the constraint of binding site independence) also failed to produce any detectable improvements in the quality of the fit.…”

“…Simulation of repeated fits showed that our data, with the HJCFIT algorithm, are capable of making good estimates of all 14 free rate constants in the flip mechanism, including the opening rate of the fully liganded receptor, ␤ 3 , which, at approximately 130,000 s Ϫ1 , is approximately twice as fast as for a nicotinic receptor (Salamone et al, 1999;Hatton et al, 2003).…”

“…In one form or another, such shut states (often referred to as "short-lived desensitized" states) have been found to be an essential feature of activation mechanisms for several receptors in this superfamily. The simplest form, used for the description of single-channel behavior for the muscle nicotinic receptor (Salamone et al, 1999;Hatton et al, 2003), involves one distal shut state (connected to the fully liganded open or shut state). The more general form in this family of mechanisms is that developed by Jones and Westbrook (1995) to explain the macroscopic behavior of GABA A receptors in response to paired short pulses of agonists and incorporates one extra shut state at each level of ligation.…”

Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating

“…There are a few lines of experimental evidence showing the contribution of M1 to channel gating (Lo et al 1991, Engel et al 1996, Croxen et al 2002, Hatton et al 2003. Our study identifies position 15?…”

“…A mutation in oM1 has been also shown to be the cause of a congenital myasthenic syndrome mainly due to an increased affinity of ACh for the resting AChR (Croxen et al . 2002, Hatton et al . 2003.…”